Fluorescent small molecules are central in many modern biological techniques. Their preparation often relies on inefficient condensation reactions requiring harsh reaction conditions with poor substrate scope. Thus, there is a need for new methods to prepare fluorophores for modern applications.
Near-infrared (near-IR, 650-900 nm) fluorophores find increasing use for a variety of techniques due to the low autofluorescence in this range. Near-IR fluorophores are uniquely well suited for in vivo fluorescence imaging due to improved tissue penetration of near-IR compared to shorter wavelengths. Heptamethine cyanines, with emission maxima around 800 nm, are perhaps the archetype (Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626-634; Alford et al., Molecular Imaging 2009, 8, 341-354). While useful in a variety of applications, including for certain clinical diagnostic procedures, many suffer from chemical stability issues at C4′ and challenging synthesis. Cyanines modified at the C4′ position with an O-alkyl substituent are desirable because these are likely to be quite stable and there is potential for a concise route to symmetrical bioconjugatable variants. However, such molecules have only rarely been described and are unknown when functionalized for biomolecule conjugation. The scarcity of C4′-O-alkyl ether cyanines is based on the inability of most alkoxide nucleophiles to undergo the standard preparative reaction, C4′-chloride exchange (Strekowski et al., J. Org. Chem. 1992, 57, 4578-4580). This failure likely stems from the poor kinetics of alkoxides in the proposed electron transfer SRN1 pathway, and competitive addition to the imine-like C2 position has been reported to intercede (Strekowski et al., J. Org. Chem. 1992, 57, 4578-4580; Strekowski et al., Dyes Pigments 2000, 46, 163-168). Thus, a need exists for stable, near-IR heptamethine cyanine fluorophores and a synthetic method for making the fluorophores.